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Myo-inositol-1-phosphate synthase (MIPS) catalyzes the NAD+-dependent isomerization of glucose-6-phosphate (G6P) into inositol-1-phosphate (IMP), controlling the rate-limiting step of the inositol pathway. Previous structural studies focused on the detailed molecular mechanism, neglecting large-scale conformational changes that drive the function of this 240 kDa homotetrameric complex. In this study, we identified the active, endogenous MIPS in cell extracts from the thermophilic fungus Thermochaetoides thermophila. By resolving the native structure at 2.48 Å (FSC = 0.143), we revealed a fully populated active site. Utilizing 3D variability analysis, we uncovered conformational states of MIPS, enabling us to directly visualize an order-to-disorder transition at its catalytic center. An acyclic intermediate of G6P occupied the active site in two out of the three conformational states, indicating a catalytic mechanism where electrostatic stabilization of high-energy intermediates plays a crucial role. Examination of all isomerases with known structures revealed similar fluctuations in secondary structure within their active sites. Based on these findings, we established a conformational selection model that governs substrate binding and eventually inositol availability. In particular, the ground state of MIPS demonstrates structural configurations regardless of substrate binding, a pattern observed across various isomerases. These findings contribute to the understanding of MIPS structure-based function, serving as a template for future studies targeting regulation and potential therapeutic applications. [ABSTRACT FROM AUTHOR]

Tyramine (TRM) is a biogenic catecholamine neurotransmitter, which can trigger migraines and hypertension. TRM accumulated in foods is reduced and detected using additive cyclodextrins (CDs) while their association characteristics remain unclear. Here, single-crystal X-ray diffraction and density functional theory (DFT) calculation have been performed, demonstrating the elusive pseudopolymorphs in β-CD inclusion complexes with TRM base/HCl, β-CD·0.5TRM·7.6H2O (1) and β-CD·TRM HCl·4H2O (2) and the rare α-CD·0.5(TRM HCl)·10H2O (3) exclusion complex. Both 1 and 2 share the common inclusion mode with similar TRM structures in the round and elliptical β-CD cavities, belong to the monoclinic space group P21, and have similar herringbone packing structures. Furthermore, 3 differs from 2, as the smaller twofold symmetry-related, round α-CD prefers an exclusion complex with the twofold disordered TRM–H+ sites. In the orthorhombic P21212 lattice, α-CDs are packed in a channel-type structure, where the column-like cavity is occupied by disordered water sites. DFT results indicate that β-CD remains elliptical to suitably accommodate TRM, yielding an energetically favorable inclusion complex, which is significantly contributed by the β-CD deformation, and the inclusion complex of α-CD with the TRM aminoethyl side chain is also energetically favorable compared to the exclusion mode. This study suggests the CD implications for food safety and drug/bioactive formulation and delivery. [ABSTRACT FROM AUTHOR]

Self‐folding cavitands represent the quintessential form of bioinspired synthetic receptors, featuring deep hydrophobic cavities that engage in host‐guest chemistry reminiscent of that operating in biomolecules. Although remarkable proof‐of‐concept applications have been reported, the narrow and rigid spaces of the legacy resorcin[4]arene derived hosts constitute a liability towards the development of specific applications in catalysis, sensing or sequestration. While notable efforts to expand the size of the cavities have been reported, the development of confined spaces reproducing the highly adaptable nature of biological receptors is a largely unaddressed issue. This review summarizes the development of a new family of calix[5]arene derived self‐folding cavitands displaying enhanced induced fit and conformational selection phenomena. Our approach capitalizes on hydrogen bonding preorganization rather than the covalent restriction approaches customary of conventional supramolecular chemistry. [ABSTRACT FROM AUTHOR]

Quadruplex‐duplex (QD) junctions, which represent unique structural motifs of both biological and technological significance, have been shown to constitute high‐affinity binding sites for various ligands. A QD hybrid construct based on a human telomeric sequence, which harbors a duplex stem‐loop in place of a short lateral loop, is structurally characterized by NMR. It folds into two major species with a (3+1) hybrid and a chair‐type (2+2) antiparallel quadruplex domain coexisting in a K+ buffer solution. The antiparallel species is stabilized by an unusual capping structure involving a thymine and protonated adenine base AH+ of the lateral loop facing the hairpin duplex to form a T·AH+·G·C quartet with the interfacial G·C base pair at neutral pH. Addition and binding of Phen‐DC3 to the QD hybrid mixture by its partial intercalation at corresponding QD junctions leads to a topological transition with exclusive formation of the (3+1) hybrid fold. In agreement with the available experimental data, such an unprecedented discrimination of QD junctions by a ligand can be rationalized following an induced fit mechanism. [ABSTRACT FROM AUTHOR]

Over the past forty years there has been a drastic increase in fructose‐related diseases, including obesity, heart disease and diabetes. Ketohexokinase (KHK), the first enzyme in the liver fructolysis pathway, catalyzes the ATP‐dependent phosphorylation of fructose to fructose 1‐phosphate. Understanding the role of KHK in disease‐related processes is crucial for the management and prevention of this growing epidemic. Molecular insight into the structure–function relationship in ligand binding and catalysis by KHK is needed for the design of therapeutic inhibitory ligands. Ketohexokinase has two isoforms: ketohexokinase A (KHK‐A) is produced ubiquitously at low levels, whereas ketohexokinase C (KHK‐C) is found at much higher levels, specifically in the liver, kidneys and intestines. Structures of the unliganded and liganded human isoforms KHK‐A and KHK‐C are known, as well as structures of unliganded and inhibitor‐bound mouse KHK‐C (mKHK‐C), which shares 90% sequence identity with human KHK‐C. Here, a high‐resolution X‐ray crystal structure of mKHK‐C refined to 1.79 Å resolution is presented. The structure was determined in a complex with both the substrate fructose and the product of catalysis, ADP, providing a view of the Michaelis‐like complex of the mouse ortholog. Comparison to unliganded structures suggests that KHK undergoes a conformational change upon binding of substrates that places the enzyme in a catalytically competent form in which the β‐sheet domain from one subunit rotates by 16.2°, acting as a lid for the opposing active site. Similar kinetic parameters were calculated for the mouse and human enzymes and indicate that mice may be a suitable animal model for the study of fructose‐related diseases. Knowledge of the similarity between the mouse and human enzymes is important for understanding preclinical efforts towards targeting this enzyme, and this ground‐state, Michaelis‐like complex suggests that a conformational change plays a role in the catalytic function of KHK‐C. [ABSTRACT FROM AUTHOR]

Riboswitches are messenger RNA (mRNA) fragments binding specific small molecules to regulate gene expression. A synthetic N1 riboswitch, inserted into yeast mRNA controls the translation of a reporter gene in response to neomycin. However, its regulatory activity is sensitive to single-point RNA mutations, even those distant from the neomycin binding site. While the association paths of neomycin to N1 and its variants remain unknown, recent fluorescence kinetic experiments indicate a two-step process driven by conformational selection. This raises the question of which step is affected by mutations. To address this, we performed all-atom twodimensional replica-exchange molecular dynamics simulations for N1 and U14C, U14C+, U15A, and A17G mutants, ensuring extensive conformational sampling of both RNA and neomycin. The obtained neomycin association and binding paths, along with multidimensional free-energy profiles, revealed a two-step binding mechanism, consisting of conformational selection and induced fit. Neomycin binds to a preformed N1 conformation upon identifying a stable upper stem and U-turn motif in the riboswitch hairpin. However, the positioning of neomycin in the binding site occurs at different RNA-neomycin distances for each mutant, which may explain their different regulatory activities. The subsequent induced fit arises from the interactions of the neomycin's N3 amino group with RNA, causing the G9 backbone to rearrange. In the A17G mutant, the critical C6-A17/G17 stacking forms at a closer RNA-neomycin distance compared to N1. These findings together with estimated binding free energies coincide with experiments and elucidate why the A17G mutation decreases and U15A enhances N1 activity in response to neomycin. [ABSTRACT FROM AUTHOR]

Abstract Quadruplex‐duplex (QD) junctions, which represent unique structural motifs of both biological and technological significance, have been shown to constitute high‐affinity binding sites for various ligands. A QD hybrid construct based on a human telomeric sequence, which harbors a duplex stem‐loop in place of a short lateral loop, is structurally characterized by NMR. It folds into two major species with a (3+1) hybrid and a chair‐type (2+2) antiparallel quadruplex domain coexisting in a K+ buffer solution. The antiparallel species is stabilized by an unusual capping structure involving a thymine and protonated adenine base AH+ of the lateral loop facing the hairpin duplex to form a T·AH+·G·C quartet with the interfacial G·C base pair at neutral pH. Addition and binding of Phen‐DC3 to the QD hybrid mixture by its partial intercalation at corresponding QD junctions leads to a topological transition with exclusive formation of the (3+1) hybrid fold. In agreement with the available experimental data, such an unprecedented discrimination of QD junctions by a ligand can be rationalized following an induced fit mechanism.

The RAS isoforms (KRAS, HRAS and NRAS) have distinct cancer type-specific profiles. NRAS mutations are the second most prevalent RAS mutations in skin and hematological malignancies. Although RAS proteins were considered undruggable for decades, isoform and mutation-specific investigations have produced successful RAS inhibitors that are either specific to certain mutants, isoforms (pan-KRAS) or target all RAS proteins (pan-RAS). While extensive structural and biochemical investigations have focused mainly on K- and H-RAS mutations, NRAS mutations have received less attention, and the most prevalent NRAS mutations in human cancers, Q61K and Q61R, are rare in K- and H-RAS. This manuscript presents a crystal structure of the NRAS Q61K mutant in the GTP-bound form. Our structure reveals a previously unseen pocket near switch II induced by the binding of a ligand to the active form of the protein. This observation reveals a binding site that can potentially be exploited for development of inhibitors against mutant NRAS. Furthermore, the well-resolved catalytic site of this GTPase bound to native GTP provides insight into the stalled GTP hydrolysis observed for NRAS-Q61K.

Protein homodimers have been classified as three-state or two-state dimers depending on whether a folded monomer forms before association, but the details of the folding–binding mechanisms are poorly understood. Kinetic transition networks of conformational states have provided insight into the folding mechanisms of monomeric proteins, but extending such a network to two protein chains is challenging as all the relative positions and orientations of the chains need to be included, greatly increasing the number of degrees of freedom. Here, we present a simplification of the problem by grouping all states of the two chains into two layers: a dissociated and an associated layer. We combined our two-layer approach with the Wako–Saito–Muñoz–Eaton method and used Transition Path Theory to investigate the dimer formation kinetics of eight homodimers. The analysis reveals a remarkable diversity of dimer formation mechanisms. Induced folding, conformational selection, and rigid docking are often simultaneously at work, and their contribution depends on the protein concentration. Pre-folded structural elements are always present at the moment of association, and asymmetric binding mechanisms are common. Our two-layer network approach can be combined with various methods that generate discrete states, yielding new insights into the kinetics and pathways of flexible binding processes. [ABSTRACT FROM AUTHOR]

Binding affinity is a fundamental parameter in drug design, describing the strength of the interaction between a molecule and its target protein. Accurately predicting binding affinity is crucial for the rapid development of novel therapeutics, the prioritization of promising candidates, and the optimization of their properties through rational design strategies. Binding affinity is determined by the mechanism of recognition between proteins and ligands. Various models, including the lock and key, induced fit, and conformational selection, have been proposed to explain this recognition process. However, current computational strategies to predict binding affinity, which are based on these models, have yet to produce satisfactory results. This article explores the connection between binding affinity and these protein-ligand interaction models, highlighting that they offer an incomplete picture of the mechanism governing binding affinity. Specifically, current models primarily center on the binding of the ligand and do not address its dissociation. In this context, the concept of ligand trapping is introduced, which models the mechanisms of dissociation. When combined with the current models, this concept can provide a unified theoretical framework that may allow for the accurate determination of the ligands’ binding affinity.

Using Anticalin technology, a lipocalin protein dubbed Colchicalin, with the ability to bind the toxic plant alkaloid colchicine with picomolar affinity, has previously been engineered, thus offering a potential antidote in vivo and also allowing its sensitive detection in biological samples. To further analyze the mode of ligand recognition, the crystal structure of Colchicalin is now reported in its unliganded form and is compared with the colchicine complex. A superposition of the protein structures revealed major rearrangements in the four structurally variable loops of the engineered lipocalin. Notably, the binding pocket in the unbound protein is largely occupied by the inward‐bent loop #3, in particular Ile97, as well as by the phenylalanine side chain at position 71 in loop #2. Upon binding of colchicine, a dramatic shift of loop #3 by up to 11.1 Å occurs, in combination with a side‐chain flip of Phe71, thus liberating the necessary space within the ligand pocket. Interestingly, the proline residue at the neighboring position 72, which arose during the combinatorial engineering of Colchicalin, remained in a cis configuration in both structures. These findings provide a striking example of a conformational adaptation mechanism, which is a long‐known phenomenon for antibodies in immunochemistry, during the recognition of a small ligand by an engineered lipocalin, thus illustrating the general similarity between the mode of antigen/ligand binding by immunoglobulins and lipocalins. [ABSTRACT FROM AUTHOR]

Receptors signal by switching between resting (C) and active (O) shapes (‘gating’) under the influence of agonists. The receptor’s maximum response depends on the difference in agonist binding energy, O minus C. In nicotinic receptors, efficiency (η) represents the fraction of agonist binding energy applied to a local rearrangement (an induced fit) that initiates gating. In this receptor, free energy changes in gating and binding can be interchanged by the conversion factor η. Efficiencies estimated from concentration-response curves (23 agonists, 53 mutations) sort into five discrete classes (%): 0.56 (17), 0.51(32), 0.45(13), 0.41(26), and 0.31(12), implying that there are 5 C versus O binding site structural pairs. Within each class efficacy and affinity are corelated linearly, but multiple classes hide this relationship. η unites agonist binding with receptor gating and calibrates one link in a chain of coupled domain rearrangements that comprises the allosteric transition of the protein.

• hNa v 1.9_C4 chimera in CHOK-1 cells encompasses the extracellular and transmembrane domain of hNa v 1.9. • Synthetic cannabinoid ACEA blocked hNa v 1.9_C4+ß1 currents causing a hyperpolarizing shift in inactivation. • hNa v 1.9_C4 channels display window currents, significantly reduced by ACEA. • Computationally, ACEA binds the local anaesthetic binding site. • The local anaesthetic profile of ACEA may be involved in cannabinoid analgesia. Cannabinoids regulate analgesia, which has aroused much interest in identifying new pharmacological therapies in the management of refractory pain. Voltage-gated Na+ channels (Na v s) play an important role in inflammatory and neuropathic pain. In particular, Na v 1.9 is involved in nociception and the understanding of its pharmacology has lagged behind because it is difficult to express in heterologous systems. Here, we utilized the chimeric channel hNa v 1.9_C4, that comprises the extracellular and transmembrane domains of hNa v 1.9, co-expressed with the ß1 subunit on CHO-K1 cells to characterize the electrophysiological effects of ACEA, a synthetic surrogate of the endogenous cannabinoid anandamide. ACEA induced a tonic block, decelerated the fast inactivation, markedly shifted steady-state inactivation in the hyperpolarized direction, decreasing the window current and showed use-dependent block, with a high affinity for the inactivated state (k i = 0.84 µM). Thus, we argue that ACEA possess a local anaesthetic-like profile. To provide a mechanistic understanding of its mode of action at the molecular level, we combined induced fit docking with Monte Carlo simulations and electrostatic complementarity. In agreement with the experimental evidence, our computer simulations revealed that ACEA binds Tyr1599 of the local anaesthetics binding site of the hNa v 1.9, contacting residues that bind cannabinol (CBD) in the Na v Ms channel. ACEA adopted a conformation remarkably similar to the crystallographic conformation of anandamide on a non-homologous protein, obstructing the Na+ permeation pathway below the selectivity filter to occupy a highly conserved binding pocket at the intracellular side. These results describe a mechanism of action, possibly involved in cannabinoid analgesia. [ABSTRACT FROM AUTHOR]

Binding kinetics has become a popular topic in pharmacology due to its potential contribution to the selectivity and duration of drug action. Yet, the overall kinetic aspects of complex binding mechanisms are still merely described in terms of elaborate algebraic equations. Interestingly, it has been recommended some 10 years ago to examine such mechanisms in terms of binding fluxes instead of the conventional rate constants. Alike the velocity of product formation in enzymology, those fluxes refer to the velocity by which one target species converts into another one. Novel binding flux‐based approaches are utilized to get a better visual insight into the "competition" between two drugs/ligands for a single target as well as between induced fit‐ and conformational selection pathways for a single ligand within a thermodynamic cycle. The present data were obtained by differential equation‐based simulations. Early on, the ligand‐binding steps "race" to equilibrium (i.e., when their forward and reverse fluxes are equal) at their individual pace. The overall/global equilibrium is only reached later on. For the competition association assays, this parting might produce a transient "overshoot" of one of the bound target species. A similar overshoot may also show up within a thermodynamic cycle and, at first glance, suggest that the induced fit pathway dominates. Yet, present findings show that under certain circumstances, it could rather be the other way round. Novel binding flux‐based approaches offer visually attractive insights into crucial aspects of "complex" binding mechanisms under non‐equilibrium conditions. [ABSTRACT FROM AUTHOR]

Prodigiosin, a red linear tripyrrole pigment, is a typical secondary metabolite with numerous biological functions, such as anticancer, antibacterial and immunosuppressant activities, and is synthesized through a bifurcated biosynthesis pathway from 4-methoxy-2,2′-bipyrrole-5-carbaldehyde (MBC) and 2-methyl-3-n-amylpyrrole (MAP). The last step in the biosynthetic pathway of MBC is catalysed by PigF, which transfers a methyl group to 4-hydroxy-2,20-bipyrrole-5-carbaldehyde (HBC) to form the final product MBC. However, the catalytic mechanism of PigF is still elusive. In this study, crystal structures of apo PigF and S-adenosylhomocysteine (SAH)-bound PigF were determined. PigF forms a homodimer and each monomer consists of two domains: a C-terminal catalytic domain and an N-terminal dimerization domain. Apo PigF adopts an open conformation, while the structure of the complex with the product SAH adopts a closed conformation. The binding of SAH induces dramatic conformational changes of PigF, suggesting an induced-fit substrate-binding mechanism. Further structural comparison suggests that this induced-fit substrate-recognition mechanism may generally exist in O-methyltransferases. Docking and mutation studies identified three key residues (His98, His247 and Asp248) that are crucial for enzyme activity. The essential function of His247 and Asp248 and structure analysis suggests that both residues are involved in activation of the HBC substrate of PigF. The invariance of Asp248 in PigF further confirmed its essential role. The invariance and essential role of His98 in PigF suggests that it is involved in correctly positioning the substrate. This study provides new insight into the catalytic mechanism of PigF, reveals an induced-fit substrate-recognition model for PigF and broadens the understanding of O-methyltransferases.

[Display omitted] • Cryptic sites potentially expand the space of druggable proteins. • Cryptic sites formed solely by side chain motion don't provide nanomolar ligand binding affinity. • However, cryptic sites formed by loop and domain motion can be valuable drug targets. • The difference in affinity is due to the much shorter time scale of side chain motion. • The different behavior is explained in terms of inhibition models and the kinetics of binding. Cryptic sites can expand the space of druggable proteins, but the potential usefulness of such sites needs to be investigated before any major effort. Given that the binding pockets are not formed, the druggability of such sites is not well understood. The analysis of proteins and their ligands shows that cryptic sites that are formed primarily by the motion of side chains moving out of the pocket to enable ligand binding generally do not bind drug-sized molecules with sufficient potency. By contrast, sites that are formed by loop or hinge motion are potentially valuable drug targets. Arguments are provided to explain the underlying causes in terms of classical enzyme inhibition theory and the kinetics of side chain motion and ligand binding. [ABSTRACT FROM AUTHOR]

Protein homodimers have been classified as three-state or two-state dimers depending on whether a folded monomer forms before association, but the details of the folding–binding mechanisms are poorly understood. Kinetic transition networks of conformational states have provided insight into the folding mechanisms of monomeric proteins, but extending such a network to two protein chains is challenging as all the relative positions and orientations of the chains need to be included, greatly increasing the number of degrees of freedom. Here, we present a simplification of the problem by grouping all states of the two chains into two layers: a dissociated and an associated layer. We combined our two-layer approach with the Wako–Saito–Muñoz–Eaton method and used Transition Path Theory to investigate the dimer formation kinetics of eight homodimers. The analysis reveals a remarkable diversity of dimer formation mechanisms. Induced folding, conformational selection, and rigid docking are often simultaneously at work, and their contribution depends on the protein concentration. Pre-folded structural elements are always present at the moment of association, and asymmetric binding mechanisms are common. Our two-layer network approach can be combined with various methods that generate discrete states, yielding new insights into the kinetics and pathways of flexible binding processes.

How proteins switch between various ligand-free and ligand-bound structures has been a key biophysical question ever since the postulation of the Monod-Wyman-Changeux and Koshland-Nemethy-Filmer models over six decades ago. The ability of NMR spectroscopy to provide structural and kinetic information on biomolecular conformational exchange places it in a unique position as an analytical tool to interrogate the mechanisms of biological processes such as protein folding and biomolecular complex formation. In addition, recent methodological developments in the areas of saturation transfer and relaxation dispersion have expanded the scope of NMR for probing the mechanics of transitions in systems where one or more states constituting the exchange process are sparsely populated and 'invisible' in NMR spectra. In this review, we highlight some of the strategies available from NMR spectroscopy for examining the nature of multi-site conformational exchange, using five case studies that have employed NMR, either in isolation, or in conjunction with other biophysical tools.

The RAS isoforms (KRAS, HRAS and NRAS) have distinct cancer type-specific profiles. NRAS mutations are the second most prevalent RAS mutations in skin and hematological malignancies. Although RAS proteins were considered undruggable for decades, isoform and mutation-specific investigations have produced successful RAS inhibitors that are either specific to certain mutants, isoforms (pan-KRAS) or target all RAS proteins (pan-RAS). While extensive structural and biochemical investigations have focused mainly on K- and H-RAS mutations, NRAS mutations have received less attention, and the most prevalent NRAS mutations in human cancers, Q61K and Q61R, are rare in K- and H-RAS. This manuscript presents a crystal structure of the NRAS Q61K mutant in the GTP-bound form. Our structure reveals a previously unseen pocket near switch II induced by the binding of a ligand to the active form of the protein. This observation reveals a binding site that can potentially be exploited for development of inhibitors against mutant NRAS. Furthermore, the well-resolved catalytic site of this GTPase bound to native GTP provides insight into the stalled GTP hydrolysis observed for NRAS-Q61K. • NRAS codon 61 mutations are prevalent in skin and hematological malignancies • NRAS structure/function has been the least studied among the RAS isoforms. • Solved crystal structure of activated NRAS Q61K mutant bound to native GTP. • Structure reveals ligand-induced pocket near switch II not previously seen in NRAS. • High-resolution structure provides insight into severe catalytic impairment of Q61K. [ABSTRACT FROM AUTHOR]

6‐Phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) catalyses the oxidative decarboxylation of 6‐phosphogluconate to ribulose 5‐phosphate in the context of the oxidative part of the pentose phosphate pathway. Depending on the species, it can be a homodimer or a homotetramer. Oligomerization plays a functional role not only because the active site is at the interface between subunits but also due to the interlocking tail‐modulating activity, similar to that of isocitrate dehydrogenase and malic enzyme, which catalyse a similar type of reaction. Since the pioneering crystal structure of sheep liver 6PGDH, which allowed motifs common to the β‐hydroxyacid dehydrogenase superfamily to be recognized, several other 6PGDH crystal structures have been solved, including those of ternary complexes. These showed that more than one conformation exists, as had been suggested for many years from enzyme studies in solution. It is inferred that an asymmetrical conformation with a rearrangement of one of the two subunits underlies the homotropic cooperativity. There has been particular interest in the presence or absence of sulfate during crystallization. This might be related to the fact that this ion, which is a competitive inhibitor that binds in the active site, can induce the same 6PGDH configuration as in the complexes with physiological ligands. Mutagenesis, inhibitors, kinetic and binding studies, post‐translational modifications and research on the enzyme in cancer cells have been complementary to the crystallographic studies. Computational modelling and new structural studies will probably help to refine the understanding of the functioning of this enzyme, which represents a promising therapeutic target in immunity, cancer and infective diseases. 6PGDH also has applied‐science potential as a biosensor or a biobattery. To this end, the enzyme has been efficiently immobilized on specific polymers and nanoparticles. This review spans the 6PGDH literature and all of the 6PGDH crystal structure data files held by the Protein Data Bank. [ABSTRACT FROM AUTHOR]

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. After its release from presynaptic nerve terminals, glutamate is quickly removed from the synaptic cleft by excitatory amino acid transporters (EAATs) 1–5, a subfamily of glutamate transporters. The five proteins utilize a complex transport stoichiometry that couples glutamate transport to the symport of three Na+ ions and one H+ in exchange with one K+ to accumulate glutamate against up to 106-fold concentration gradients. They are also anion-selective channels that open and close during transitions along the glutamate transport cycle. EAATs belong to a larger family of secondary-active transporters, the SLC1 family, which also includes purely Na+- or H+-coupled prokaryotic transporters and Na+-dependent neutral amino acid exchangers. In recent years, molecular cloning, heterologous expression, cellular electrophysiology, fluorescence spectroscopy, structural approaches, and molecular simulations have uncovered the molecular mechanisms of coupled transport, substrate selectivity, and anion conduction in EAAT glutamate transporters. Here we review recent findings on EAAT transport mechanisms, with special emphasis on the highly conserved hairpin 2 gate, which has emerged as the central processing unit in many of these functions. [ABSTRACT FROM AUTHOR]

Two mechanisms, induced fit (IF) and conformational selection (CS), have been proposed to explain ligand recognition coupled conformational changes. The histidine binding protein (HisJ) adopts the CS mechanism, in which a pre-equilibrium is established between the open and the closed states with the ligand binding to the closed state. Despite being structurally similar to HisJ, the maltose binding protein (MBP) adopts the IF mechanism, in which the ligand binds the open state and induces a transition to the closed state. To understand the molecular determinants of this difference, we performed molecular dynamics (MD) simulations of coarse-grained dual structure based models. We find that intra-protein contacts unique to the closed state are sufficient to promote the conformational transition in HisJ, indicating a CS-like mechanism. In contrast, additional ligand-mimicking contacts are required to “induce” the conformational transition in MBP suggesting an IF-like mechanism. In agreement with experiments, destabilizing modifications to two structural features, the spine helix (SH) and the balancing interface (BI), present in MBP but absent in HisJ, reduce the need for ligand-mimicking contacts indicating that SH and BI act as structural restraints that keep MBP in the open state. We introduce an SH like element into HisJ and observe that this can impede the conformational transition increasing the importance of ligand-mimicking contacts. Similarly, simultaneous mutations to BI and SH in MBP reduce the barrier to conformational transitions significantly and promote a CS-like mechanism. Together, our results show that structural restraints present in the protein structure can determine the mechanism of conformational transitions and even simple models that correctly capture such structural features can predict their positions. MD simulations of such models can thus be used, in conjunction with mutational experiments, to regulate protein ligand interactions, and modulate ligand binding affinities.

Binding affinity is a fundamental parameter in drug design, describing the strength of the interaction between a molecule and its target protein. Accurately predicting binding affinity is crucial for the rapid development of novel therapeutics, the prioritization of promising candidates, and the optimization of their properties through rational design strategies. Binding affinity is determined by the mechanism of recognition between proteins and ligands. Various models, including the lock and key, induced fit, and conformational selection, have been proposed to explain this recognition process. However, current computational strategies to predict binding affinity, which are based on these models, have yet to produce satisfactory results. This article explores the connection between binding affinity and these protein-ligand interaction models, highlighting that they offer an incomplete picture of the mechanism governing binding affinity. Specifically, current models primarily center on the binding of the ligand and do not address its dissociation. In this context, the concept of ligand trapping is introduced, which models the mechanisms of dissociation. When combined with the current models, this concept can provide a unified theoretical framework that may allow for the accurate determination of the ligands' binding affinity.

Induced fit- (IF) and conformational selection (CS) binding mechanisms have long been regarded to be mutually exclusive. Yet, they are now increasingly considered to produce the final ligand-target complex alongside within a thermodynamic cycle. This viewpoint benefited from the introduction of binding fluxes as a tool for analyzing the overall behavior of such cycle. This study aims to provide more vivid and applicable insights into this emerging field. In this respect, combining differential equation-based simulations and hitherto little explored alternative modes of calculation provide concordant information about the intricate workings of such cycle. In line with previous reports, we observe that the relative contribution of IF increases with the ligand concentration at equilibrium. Yet the baseline contribution may vary from one case to another and simulations as well as calculations show that this parameter is essentially regulated by the dissociation rate of both pathways. Closer attention should be paid to how the contributions of IF and CS compare at physiologically relevant drug/ligand concentrations. To this end, a simple equation discloses how changing a limited set of “microscopic” rate constants can extend the concentration range at which CS contributes most effectively. Finally, it could also be beneficial to extend the utilization of flux- based approaches to more physiologically relevant time scales and alternative binding models. [ABSTRACT FROM AUTHOR]

Rediscoveries are not rare in biology. A recent example is the re-birth of the "fluctuation fit" concept developed by F. B. Straub and G. Szabolcsi in the sixties of the last century, under various names, the most popular of which is the "conformational selection". This theory offers an alternative to the "induced fit" concept by Koshland for the interpretation of the mechanism of protein—ligand interactions. A central question is whether the ligand induces a conformational change (as described by the induced fit model) or rather selects and stabilizes a complementary conformation from a pre-existing equilibrium of various states of the protein (according to the fluctuation fit/conformational selection model). Straub and Szabolcsi's role and the factors hindering the spread of the fluctuation fit theory are discussed in the context of the history of the Hungarian biology in the 1950s and 1960s. [ABSTRACT FROM AUTHOR]

Abstract Induced fit‐ (IF) and conformational selection (CS) binding mechanisms have long been regarded to be mutually exclusive. Yet, they are now increasingly considered to produce the final ligand‐target complex alongside within a thermodynamic cycle. This viewpoint benefited from the introduction of binding fluxes as a tool for analyzing the overall behavior of such cycle. This study aims to provide more vivid and applicable insights into this emerging field. In this respect, combining differential equation‐ based simulations and hitherto little explored alternative modes of calculation provide concordant information about the intricate workings of such cycle. In line with previous reports, we observe that the relative contribution of IF increases with the ligand concentration at equilibrium. Yet the baseline contribution may vary from one case to another and simulations as well as calculations show that this parameter is essentially regulated by the dissociation rate of both pathways. Closer attention should be paid to how the contributions of IF and CS compare at physiologically relevant drug/ligand concentrations. To this end, a simple equation discloses how changing a limited set of “microscopic” rate constants can extend the concentration range at which CS contributes most effectively. Finally, it could also be beneficial to extend the utilization of flux‐ based approaches to more physiologically relevant time scales and alternative binding models.

This report describes the implementation of a laboratory exercise for an advanced biochemistry or enzyme kinetics class at the undergraduate or graduate level, designed to improve understanding of protein conformational changes associated with the binding of a ligand. Students measure the fluorescence changes induced by the conformational transition of a glycoprotein (the Na,K-ATPase) upon addition of different ligands (Pi and BeF3−) and analyse the results in order to determine the mechanism of the process. The results show that Pi and BeF3− present opposite effects on the observed rate constants (kobs) with ligand concentration: kobs decreases with [Pi] and increases with [BeF3−]. This observation, together with the frequently used assumption that binding occurs under rapid equilibrium, led to propose different models for ligand-induced conformational transitions: a conformational selection for Pi and an induced fit for BeF3−. In this paper, we show that if the rapid-equilibrium approximation for ligand binding is not assumed, a conformational selection mechanism can account for the effects of both ligands. This active-learning exercise serves as the basis for discussing the consequences of not being extremely cautious when invoking approximations about not-very-well-known systems and the importance of a correct understanding of models assigned to chemical processes. [ABSTRACT FROM AUTHOR]

Prolidase (EC 3.4.13.9) is an enzyme that specifically hydrolyzes Xaa-Pro dipeptides into free amino acids. We previously studied kinetic behaviours and solved the crystal structure of wild-type (WT) Lactococcus lactis prolidase (Ll prol), showing that this homodimeric enzyme has unique characteristics: allosteric behaviour and substrate inhibition. In this study, we focused on solving the crystal structures of three Ll prol mutants (D36S, H38S, and R293S) which behave differently in v - S plots. The D36S and R293S Ll prol mutants do not show allosteric behaviour, and the Ll prol mutant H38S has allosteric behaviour comparable to the WT enzyme (Hill constant 1.52 and 1.58, respectively). The crystal structures of Ll prol variants suggest that the active site of Ll prol formed with amino acid residues from both monomers, i.e. , located in an interfacial area of dimer. The comparison between the structure models of Ll prol indicated that the two monomers in the dimers of Ll prol variants have different relative positions among Ll prol variants. They showed different interatomic distances between the amino acid residues bridging the two monomers and varied sizes of the solvent-accessible interface areas in each Ll prol variant. These observations indicated that Ll prol could adapt to different conformational states with distinctive substrate affinities. It is strongly speculated that the domain movements required for productive substrate binding are restrained in allosteric Ll prol (WT and H38S). At low substrate concentrations, only one out of the two active sites at the dimer interface could accept substrate; as a result, the asymmetrical activated dimer leads to allosteric behaviour. • Crystal structures of prolidases with different degrees of allostery was solved. • Flexible loop located at the homodimer interface of L. lactis prolidase. • Structure alignment reveals different conformational states of prolidase. • Intermolecular interactions at the homodimer interface are key to allostery. [ABSTRACT FROM AUTHOR]

Here we describe the X-ray crystal structure of a double-Trp mutant (Gly46→Trp/Gly262→Trp) of the lactose permease of Escherichia coli (LacY) with a bound, high-affinity lactose analog. Although thought to be arrested in an open-outward conformation, the structure is almost occluded and is partially open to the periplasmic side; the cytoplasmic side is tightly sealed. Surprisingly, the opening on the periplasmic side is sufficiently narrow that sugar cannot get in or out of the binding site. Clearly defined density for a bound sugar is observed at the apex of the almost occluded cavity in the middle of the protein, and the side chains shown to ligate the galactopyranoside strongly confirm more than two decades of biochemical and spectroscopic findings. Comparison of the current structure with a previous structure of LacY with a covalently bound inactivator suggests that the galactopyranoside must be fully ligated to induce an occluded conformation. We conclude that protonated LacY binds D-galactopyranosides specifically, inducing an occluded state that can open to either side of the membrane.

In biology, supramolecular recognition typically involves an 'induced‐fit' mechanism, where structures rearrange upon complexation to accommodate binding ligands. Designing minimalistic compounds with such adaptability is challenging as they involve subtle conformational changes that are energetically similar. Here, we demonstrate the integration of combinatorial screening with molecular modelling to identify heptapeptides that form a stable loop upon recognition of uridine triphosphate (UTP). Peptide sequences selected using phage display were refined computationally and correlated with experimental KD values. This combined approach may serve as a method for the de novo selection and subsequent rationalization of the compositional and organizational principles that dictate chemical functionality in flexible structures with dynamic conformations. [ABSTRACT FROM AUTHOR]

We used the Surface Forces Apparatus to elucidate the interaction mechanism between grafted 5 heptad-long peptides engineered to spontaneously form a heterodimeric coiled-coil complex. The results demonstrated that when intimate contact between peptides is reached, binding occurs first via weakly interacting but more mobile distal heptads, suggesting an induced-fit association process. Precise control of the distance between peptide-coated surfaces allowed to quantitatively monitor the evolution of their biding energy. The binding energy of the coiled-coil complex increased in a stepwise fashion rather than monotonically with the overlapping distance, each step corresponding to the interaction between a quantized number of heptads. Surface forces data were corroborated to surface plasmon resonance measurements and molecular dynamics simulations and allowed the calculation of the energetic contribution of each heptad within the coiled-coil complex. [ABSTRACT FROM AUTHOR]

We report here a guest‐reaction‐induced mitosis‐like host transformation from a known Pd4L2 cage 1 to a conjoined Pd6L3 twin‐cage 2 featuring two separate cavities. The encapsulation of 1‐hydroxymethyl‐2‐naphthol (G1), a known ortho‐quinone methide (o‐QMs) precursor, within the hydrophobic cavity of cage 1 is found crucial to realize the cage to twin‐cage conversion. Confined G1 molecules within the nanocavity undergo self‐coupling dimerization reaction to form 2,2′‐dihydroxy‐1,1′‐dinaphthylmethane (G2) which then triggers the cage to twin‐cage mitosis. The same conversion also proceeds, in a much faster rate, via the direct templation of G2, confirming the induced‐fit transformation mechanism. The structure of the (G2)2⊂2 host–guest complex has been established by X‐ray crystallographic study, where cis‐ to trans‐ conformational switch on one bridging ligand is revealed. [ABSTRACT FROM AUTHOR]

A decade ago, many high-affinity drugs were thought to bind to their target via an induced-fit pathway instead of conformational selection. Yet, both pathways make up part of a thermodynamic cycle, and, owing to binding flux-based approaches, it is now rather considered that they act in parallel and also that their relative contribution to the final ligand–target complex depends on the ligand concentration. Those approaches are of increasing interest, but published data still merely refer to the peculiar situation of equilibrium binding. This article draws attention to the benefit of extending those approaches to address more physiological nonequilibrium binding conditions and in vivo situations. For the presented example, they help to apprehend transient experimental manifestations of a 'conventional' thermodynamic cycle. Induced-fit and conformational selection mechanisms for ligand binding were long considered to be mutually exclusive, and most of the slowly dissociating drugs were proposed to act in an induced-fit-like manner. Yet, both pathways form a thermodynamic cycle. Inspection with the aid of binding flux-based approaches contributed to the current view that they act in unison and even that the dominance of one or the other depends on the ligand concentration. Those are now increasingly adopted and even extended. Yet, published data still refer to equilibrium binding, which is hardly met in vivo. This calls for extending those approaches to nonequilibrium situations. Current cycle-based simulations illustrate the utility of such extensions. They highlight their benefit for apprehending transient and occasionally even unexpected manifestations of complex binding mechanisms in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (MTB) caused 10.4 million cases of tuberculosis and 1.7 million deaths in 2016. The incidence of multidrug-resistant and extensively drug-resistant MTB is becoming an increasing threat to public health and the development of novel anti-MTB drugs is urgently needed. Methionyl-tRNA synthetase (MetRS) is considered to be a valuable drug target. However, structural characterization of M. tuberculosis MetRS (MtMetRS) was lacking for decades, thus hampering drug design. Here, two high-resolution crystal structures of MtMetRS are reported: the free-state structure (apo form; 1.9 Å resolution) and a structure with the intermediate product methionyl-adenylate (Met-AMP) bound (2.4 Å resolution). It was found that free-state MtMetRS adopts a previously unseen conformation that has never been observed in other MetRS homologues. The pockets for methionine and AMP are not formed in free-state MtMetRS, suggesting that it is in a nonproductive conformation. Combining these findings suggests that MtMetRS employs an induced-fit mechanism in ligand binding. By comparison with the structure of human cytosolic MetRS, additional pockets specific to MtMetRS that could be used for anti-MTB drug design were located.

Chromatin post‐translational modifications are thought to be important for epigenetic effects on gene expression. Methylation of histone N‐terminal tail lysine residues constitutes one of many such modifications, executed by families of histone lysine methyltransferase (HKMTase). One such protein is ASHH2 from the flowering plant Arabidopsis thaliana, equipped with the interaction domain, CW, and the HKMTase domain, SET. The CW domain of ASHH2 is a selective binder of monomethylation at lysine 4 on histone H3 (H3K4me1) and likely helps the enzyme dock correctly onto chromatin sites. The study of CW and related interaction domains has so far been emphasizing lock–key models, missing important aspects of histone‐tail CW interactions. We here present an analysis of the ASHH2 CW‐H3K4me1 complex using NMR and molecular dynamics, as well as mutation and affinity studies of flexible coils. β‐augmentation and rearrangement of coils coincide with changes in the flexibility of the complex, in particular the η1, η3 and C‐terminal coils, but also in the β1 and β2 strands and the C‐terminal part of the ligand. Furthermore, we show that mutating residues with outlier dynamic behaviour affect the complex binding affinity despite these not being in direct contact with the ligand. Overall, the binding process is consistent with conformational selection. We propose that this binding mechanism presents an advantage when searching for the correct post‐translational modification state among the highly modified and flexible histone tails, and also that the binding shifts the catalytic SET domain towards the nucleosome. Databases: Structural data are available in the PDB database under the accession code 6QXZ. Resonance assignments for CW42 in its apo‐ and holo‐forms are available in the BMRB database under the accession code 27251. [ABSTRACT FROM AUTHOR]

Aminoglycoside acetyltransferases (AACs) catalyze the transfer of an acetyl group between acetyl‐CoA and an aminoglycoside, producing CoA and an acetylated aminoglycoside. AAC(6′)‐Ii enzymes target the amino group linked to the 6′ C atom in an aminoglycoside. Several structures of the AAC(6′)‐Ii from Enterococcus faecium [Ef‐AAC(6′)‐Ii] have been reported to date. However, the detailed mechanism of its enzymatic function remains elusive. In this study, the crystal structure of Ef‐AAC(6′)‐Ii was determined in a novel substrate‐free form. Based on structural analysis, it is proposed that Ef‐AAC(6′)‐Ii sequentially undergoes conformational selection and induced fit for substrate binding. These results therefore provide a novel viewpoint on the mechanism of action of Ef‐AAC(6′)‐Ii. [ABSTRACT FROM AUTHOR]

The ATTRACT protein‐protein docking program has been employed to predict protein‐protein complex structures in CAPRI rounds 38‐45. For 11 out of 16 targets acceptable or better quality solutions have been submitted (~70%). It includes also several cases of peptide‐protein docking and the successful prediction of the geometry of carbohydrate‐protein interactions. The option of combining rigid body minimization and simultaneous optimization in collective degrees of freedom based on elastic network modes was employed and systematically evaluated. Application to a large benchmark set indicates a modest improvement in docking performance compared to rigid docking. Possible further improvements of the docking approach in particular at the scoring and the flexible refinement steps are discussed. [ABSTRACT FROM AUTHOR]

The formation of specific protein‐protein interactions is often a key to a protein's function. During complex formation, each protein component will undergo a change in the conformational state, for some these changes are relatively small and reside primarily at the sidechain level; however, others may display notable backbone adjustments. One of the classic problems in the protein‐docking field is to be able to a priori predict the extent of such conformational changes. In this work, we investigated three protocols to find the most suitable input structure conformations for cross‐docking, including a robust sampling approach in normal mode space. Counterintuitively, knowledge of the theoretically best combination of normal modes for unbound‐bound transitions does not always lead to the best results. We used a novel spatial partitioning library, Aether Engine (see Supplementary Materials), to efficiently search the conformational states of 56 receptor/ligand pairs, including a recent CAPRI target, in a systematic manner and selected diverse conformations as input to our automated docking server, SwarmDock, a server that allows moderate conformational adjustments during the docking process. In essence, here we present a dynamic cross‐docking protocol, which when benchmarked against the simpler approach of just docking the unbound components shows a 10% uplift in the quality of the top docking pose. [ABSTRACT FROM AUTHOR]

Detoxication, or 'drug‐metabolizing', enzymes and drug transporters exhibit remarkable substrate promiscuity and catalytic promiscuity. In contrast to substrate‐specific enzymes that participate in defined metabolic pathways, individual detoxication enzymes must cope with substrates of vast structural diversity, including previously unencountered environmental toxins. Presumably, evolution selects for a balance of 'adequate' kcat/KM values for a wide range of substrates, rather than optimizing kcat/KM for any individual substrate. However, the structural, energetic, and metabolic properties that achieve this balance, and hence optimize detoxication, are not well understood. Two features of detoxication enzymes that are frequently cited as contributions to promiscuity include the exploitation of highly reactive versatile cofactors, or cosubstrates, and a high degree of flexibility within the protein structure. This review examines these intuitive mechanisms in detail and clarifies the contributions of the classic ligand binding models 'induced fit' (IF) and 'conformational selection' (CS) to substrate promiscuity. The available literature data for drug metabolizing enzymes and transporters suggest that IF is exploited by these promiscuous detoxication enzymes, as it is with substrate‐specific enzymes, but the detoxication enzymes uniquely exploit 'IFs' to retain a wide range of substrates at their active sites. In contrast, whereas CS provides no catalytic advantage to substrate‐specific enzymes, promiscuous enzymes may uniquely exploit it to recruit a wide range of substrates. The combination of CS and IF, for recruitment and retention of substrates, can potentially optimize the promiscuity of drug metabolizing enzymes and drug transporters. [ABSTRACT FROM AUTHOR]

We describe a strategy for constructing atomic resolution dynamical ensembles of RNA molecules, spanning up to millisecond timescales, that combines molecular dynamics (MD) simulations with NMR residual dipolar couplings (RDC) measured in elongated RNA. The ensembles are generated via a Monte Carlo procedure by selecting snap-shot from an MD trajectory that reproduce experimentally measured RDCs. Using this approach, we construct ensembles for two variants of the transactivation response element (TAR) containing three (HIV-1) and two (HIV-2) nucleotide bulges. The HIV-1 TAR ensemble reveals significant mobility in bulge residues C24 and U25 and to a lesser extent U23 and neighboring helical residue A22 that give rise to large amplitude spatially correlated twisting and bending helical motions. Omission of bulge residue C24 in HIV-2 TAR leads to a significant reduction in both the local mobility in and around the bulge and amplitude of inter-helical bending motions. In contrast, twisting motions of the helices remain comparable in amplitude to HIV-1 TAR and spatial correlations between them increase significantly. Comparison of the HIV-1 TAR dynamical ensemble and ligand bound TAR conformations reveals that several features of the binding pocket and global conformation are dynamically preformed, providing support for adaptive recognition via a ‘conformational selection’ type mechanism.

Intrinsically disordered proteins (IDPs) are widespread in eukaryotes and participate in a variety of important cellular processes. Numerous studies using state-of-the-art experimental and theoretical methods have advanced our understanding of IDPs and revealed that disordered regions engage in a large repertoire of intra- and intermolecular interactions through their conformational dynamics, thereby regulating many intracellular functions in concert with folded domains. The mechanisms by which IDPs interact with their partners are diverse, depending on their conformational propensities, and include induced fit, conformational selection, and their mixtures. In addition, IDPs are implicated in many diseases, and progress has been made in designing inhibitors of IDP-mediated interactions. Here we review these recent advances with a focus on the dynamics and interactions of IDPs. [ABSTRACT FROM AUTHOR]

Ubiquitin (Ub) specifically interacts with the Ub-associating domain (UBA) in a proteasomal shuttle factor, while the latter is involved in either proteasomal targeting or self-assembly coacervation. PINK1 phosphorylates Ub at S65 and makes Ub alternate between C-terminally relaxed (pUbRL) and retracted conformations (pUbRT). Using NMR spectroscopy, we show that pUbRL but not pUbRT preferentially interacts with the UBA from two proteasomal shuttle factors Ubqln2 and Rad23A. Yet discriminatorily, Ubqln2-UBA binds to pUb more tightly than Rad23A does and selectively enriches pUbRL upon complex formation. Further, we determine the solution structure of the complex between Ubqln2-UBA and pUbRL and uncover the thermodynamic basis for the stronger interaction. NMR kinetics analysis at different timescales further suggests an indued-fit binding mechanism for pUb-UBA interaction. Notably, at a relatively low saturation level, the dissociation rate of the UBA-pUbRL complex is comparable with the exchange rate between pUbRL and pUbRT. Thus, a kinetic constraint would dictate the interaction between Ub and UBA, thus fine-tuning the functional state of the proteasomal shuttle factors.

Xeroderma pigmentosum complementation group D (XPD) is a UV-sensitive syndrome and a rare incurable genetic disease which is caused by the genetic mutation of the excision repair cross-complementation group 2 gene (ERCC2). Patients who harbor only XPD R683W mutant protein develop severe photosensitivity and progressive neurological symptoms. Cultured cells derived from patients with XPD (XPD R683W cells) demonstrate a reduced nucleotide excision repair (NER) ability. We hope to ameliorate clinical symptoms if we can identify candidate agents that would aid recovery of the cells’ NER ability. To investigate such candidates, we created in silico methods of drug repurposing (in silico DR), a strategy that utilizes the recovery of ATP-binding in the XPD R683W protein after the induced fit. We chose 4E1RCat and aprepitant as the candidates for our in silico DR, and evaluated them by using the UV-induced unscheduled DNA synthesis (UDS) assay to verify the recovery of NER in XPD R683W cells. UDS values of the cells improved about 1.4–1.7 times after 4E1RCat treatment compared with solvent-only controls; aprepitant showed no positive effect. In this study, therefore, we succeeded in finding the candidate agent 4E1RCat for XPD R683W. We also demonstrated that our in silico DR method is a cost-effective approach for drug candidate discovery.

In the design of 1‐phenylbenzimidazoles as model cyclooxygenase (COX) inhibitors, docking to a series of crystallographic COX structures was performed to evaluate their potential for high‐affinity binding and to reproduce the interaction profile of well‐known COX inhibitors. The effect of ligand‐specific induced fit on the calculations was also studied. To quantitatively compare the pattern of interactions of model compounds to the profile of several cocrystallized COX inhibitors, a geometric parameter, denominated ligand‐receptor contact distance (LRCD), was developed. The interaction profile of several model complexes showed similarity to the profile of COX complexes with inhibitors such as iodosuprofen, iodoindomethacin, diclofenac, and flurbiprofen. Shaping of high‐affinity binding sites upon ligand‐specific induced fit mostly determined both the affinity and the binding mode of the ligands in the docking calculations. The results suggest potential of 1‐phenylbenzimidazole derivatives as COX inhibitors on the basis of their predicted affinity and interaction profile to COX enzymes. The analyses also provided insights into the role of induced fit in COX enzymes. While inhibitors produce different local structural changes at the COX ligand binding site, induced fit allows inhibitors in diverse chemical classes to share characteristic interaction patterns that ensure key contacts to be achieved. Different interaction patterns may also be associated with different inhibitory mechanisms. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae pullulanase (KPP) belongs to glycoside hydrolase family 13 subfamily 13 (GH13_13) and is the only enzyme that is reported to perform an induced‐fit motion of the active‐site loop (residues 706–710). Comparison of pullulanase structures indicated that only KPP has Leu680 present behind the loop, in contrast to the glycine found in other GH13_13 members. Analysis of the structure and activity of recombinant pullulanase from K. pneumoniae ATCC 9621 (rKPP) and its mutant (rKPP‐G680L) indicated that the side chain of residue 680 is important for the induced‐fit motion of the loop 706–710 and alters the binding affinity of the substrate. [ABSTRACT FROM AUTHOR]

Porous materials that can undergo pore‐structure adjustment to better accommodate specific molecules are ideal for separation and purification. Here, we report a stable microporous metal‐organic framework, JNU‐1, featuring one‐dimensional diamond‐shaped channels with a high density of open metal sites arranged on the surface for the cooperative binding of acetylene. Together with its framework flexibility and appropriate pore geometry, JNU‐1 exhibits an induced‐fit behavior for acetylene. The specific binding sites and continuous framework adaptation upon increased acetylene pressure are validated by molecular modeling and in situ X‐ray diffraction study. This unique induced‐fit behavior endows JNU‐1 with an unprecedented increase in the acetylene binding affinity (adsorption enthalpy: up to 47.6 kJ mol−1 at ca. 2.0 mmol g−1 loading). [ABSTRACT FROM AUTHOR]